Development of reverse-transcription PCR techniques to analyse the density and sex ratio of gametocytes in genetically diverse Plasmodium chabaudi infections
Introduction
The application of molecular methods to detect and quantify different life stages of malaria (Plasmodium) parasites has provided novel insights into infection dynamics of relevance to evolutionary biology, epidemiology and medical disciplines [1]. Unlike traditional microscopy, molecular methods, including quantitative PCR (qPCR) and nucleic acid sequence-based amplification (QT-NASBA), are sensitive enough to reveal subtle variations in infection dynamics and detect parasites infections considered sub-patent by microscopy [2], [3], [4], [5], [6]. Furthermore, traditional microscopy cannot be used to follow the progress of individual genotypes (clones) in mixed infections that contain multiple distinct genotypes. Genotype-specific quantitative reverse-transcription PCR (qRT-PCR) methods have been developed for the rodent malaria, Plasmodium chabaudi, to quantify the number of parasites belonging to different genotypes within the same infection [7], [8], [9]. Application of these methods has revealed that genotypes of P. chabaudi suffer from competition with con-specific genotypes and these effects can be linked to the virulence of co-infecting genotypes [7], [8], [9], [10], [11]. Despite genetically diverse infections being the rule in nature, we are yet to examine how competition in genetically diverse infections influences sex ratios produced by individual genotypes [12].
To transmit to vectors, malaria and related parasites produce gametocytes, and these stages are either male or female. The gametocytes differentiate into gametes as soon as they are taken up in a vector's blood meal. Each male gametocyte can produce up to eight male gametes and each female gametocyte differentiates into a single female gamete. Male gametes locate and fertilise female gametes and the resulting zygotes undergo several development stages in their vectors before being ready to re-infect new vertebrate hosts [13]. Successful transmission to vectors depends on the number of gametocytes produced and the ratio of males to females (sex ratio: proportion of gametocytes that are male) [14], [15], [16]. While gametocytes can now be quantified by PCR, their sex is traditionally assigned by microscopy. This method of sexing has been shown to overestimate the proportion of female gametocytes, as maturing male gametocytes resemble females and can be incorrectly sexed [17], [18]. In addition, gametocyte densities are often extremely low which results in variable and inaccurate sex ratio estimates [19]. Furthermore, microscopy cannot be used to distinguish between male and female gametocytes produced by different genotypes in mixed-genotype infections.
To overcome these issues we developed cross-genotype quantitative reverse-transcription PCR assays for following gametocyte density, male gametocyte density and asexual parasite density throughout P. chabaudi infections. We used information derived from recent proteome analysis of male and female gametocytes in the closely related rodent malaria parasite Plasmodium berghei, which identified a large cohort of proteins which were pre-synthesised exclusively by male or female gametocytes [20] in preparation for a swift differentiation into gametes. We then extended these assays to be genotype-specific so we could follow the gametocyte density and sex ratio of individual genotypes in mixed-genotype infections. We used our assays to determine how gametocyte production and sex ratio vary throughout infections in two genetically distinct lines (genotypes) and how these traits are influenced by competition in mixed-genotype infections comprising both of these genotypes.
Section snippets
Parasites and hosts
In all experiments male MF1 mice (in house supplier, University of Edinburgh) were infected with clonal P. chabaudi lines AS, AJ or CR (WHO Registry of Standard Malaria Parasites, The University of Edinburgh). All mice were 6–8 weeks old and received an intra-peritoneal inoculation of 106 parasitized red blood cells (RBC) in a 0.1 μl dose as previously described [21]. Mice were housed at 21 °C with a 12 h light cycle, and maintained on a diet of SDS41B food pellets (Harlan Scientific, UK) and
Sensitivity and strain specificity of qRT-PCR assays
The universal total parasite qRT-PCR assay could detect a minimum density of approximately 25 parasites/μl of blood and the AJ and AS-specific assays for measuring total parasites detected approximately two-fold fewer parasites (Table 1). These sensitivities are comparable to a previous study [5], which reported a total parasite detection sensitivity of 740 parasites/μl of blood, using a qRT-PCR assay based on the measurement of MSP-1 gene copies in DNA isolated from P. chabaudi infected blood.
Discussion
We have developed qRT-PCR assays to detect and quantify total parasite, total gametocyte and male gametocyte densities throughout the course of P. chabaudi infections. The qRT-PCR assays we developed are based on the detection of mRNA transcripts of P. chabaudi homologues of the P. berghei genes PB00198.00.0 (CG1) and PB000791.00.0 (MG1). These genes were chosen because their proteins were not detected in the proteome of asexual P. berghei parasites and were strongly predicted by the proteomic
Acknowledgements
We thank the NERC, Wellcome Trust and BBSRC for funding; S. Khan, J. Thompson, A. Wargo, A. Bell and D. Nussey for help and discussion. We also thank A. Read for advice and discussion and for allowing us the use of laboratory space and equipment.
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2009, Trends in ParasitologyCitation Excerpt :A similar study examined another avian malaria parasite (Parahaemoprotus [2]) but found no relationship between prevalence and gametocyte sex ratio [33]. New molecular techniques now provide very precise determinations of gametocyte sex ratio (the equivalent of counting millions of cells) [40] and enable the number of coexisting clones [39] and even their relative proportions to be measured [41]. Unfortunately, accurately measuring male fecundity remains challenging, but estimates can be inferred from sex ratio of solitary infections (although this is a reversed use of the theory to estimate a parameter in the equations).
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